Studies in a number of laboratories, including ours, have shown that eukaryotic cells contain enzymes that transfer sugars from their nucleotide derivatives to polyisoprenol phosphate. Studying cell-free preparations of hen oviduct we have demonstrated such a reaction with GDP-mannose. Moreover, we have shown that mannosyl phosphoryl polyprenol (MPP) serves as a mannosyl donor in synthesis of an oligosaccharide-lipid and in synthesis of mannose-containing glycoproteins. Preliminary evidence indicates that the oligosaccharide- lipid is formed from MPP and that the mannose-containing oligosaccharide chain of it is transferred en bloc to the glycoproteins. We propose to isolate the oligosaccharide-lipid and to chemically characterize it. With isolated, purified oligosaccharide-lipid we hope to establish conclusively that this compound is a donor of the oligosaccharide chain in glycoprotein assembly. To aid in studying the enzymatic synthesis and structure of the oligosaccharide-lipid two approaches will be taken. One will involve synthesis of (H3)-labeled polyprenol phosphate so that oligosaccharide-lipid labeled in both the mannose residue and the lipid moiety can be prepared, and its metabolism can be studied. The second approach will involve double labeling of the oligosaccharide chain with both mannose and glucosamine, which appears to be the sugar that links the oligosaccharide to the lipid. The subcellular sites of synthesis of MPP, oligosaccharide-lipid and glycoprotein will be studied by classical cell fractionation procedures. In addition attempts to isolate suspensions of oviduct cells will be made. Using exogenous GDP-mannose labeling of the mannose-containing membrane glycoproteins (presumably plasma membrane glycoproteins) will be studied with these intact cells. If labeling is observed, these proteins will be compared with those formed in cell-free preparations. In addition attempts to determine whether or not MPP and oligosaccharide-lipid participate in the labeling of these plasma membrane glycoproteins will be studied with cell suspensions.